Cellular membrane ATP-sensitive K+ channel (hereinafter to be referred to as sarcKATP channel) is known to present in the myocardium cellular membrane, smooth muscle cellular membrane and pancreatic β cell membrane, and regulate the calcium concentration in the cytoplasm (see non-patent reference 1). Conventionally, openers of the KATP channel in the myocardium cellular membrane and smooth muscle cellular membrane have been studied as therapeutic drugs for angina pectoris and hypertension. The KATP channel blocker of the pancreatic β cellular membrane has been placed in the market as a therapeutic drug for diabetes.
In recent years, it has been pharmacologically and physiologically shown that the mitoKATP channel is present and ischemic injury of cell can be prevented by opening the channel (see non-patent reference 2).
During ischemia, in the cell, Na/K ATPase activity on the cellular membrane decreases due to the cessation of energy production, and intracellular Na concentration increases. Upon reperfusion of blood flow, intracellular Na that increased during ischemia is exchanged with extracellular Ca to increase intracellular Ca. Ca influx into mitochondria increases intramitochondrial Ca concentration and depolarizes inner mitochondrial membrane. Moreover, the reactive oxygen species produced by reperfusion also promotes depolarization of the inner mitochondrial membrane.
When the mitoKATP channel is previously opened, Ca influx into mitochondria can be inhibited during reperfusion, and depolarization of the inner mitochondrial membrane is also inhibited (see non-patent reference 3).
For study of the relationship between the mitoKATP channel opening action and pathology, (1) an evaluation method using a cell line such as Girardi cell, PC12 cell, SHSY-5Y cell, human neuroblastoma cell, primary cultured rat cardiac muscle cell and the like, (2) an evaluation method using mitochondria isolated from a cell by centrifugation, (3) a method based on the evaluation of the effect on the isolated heart and the like are generally known.
Furthermore, the following evaluation method using Griardi cells (GIRARDI HEART: ECACC No. 9312082) has been reported.    (1) Griardi cells were seeded on a 24-well culture plate, adenosine or diazoxide (mitoKATP channel opener) was added, the cells were cultured for 3 hr in an acidified culture medium containing a metabolism inhibitor and the like under low oxygen conditions to reproduce an ischemic state, and further cultured for 1 hr in a normal culture medium at a normal oxygen concentration. Thereafter, to evaluate the level of apoptosis, a cell death indicator PI (propidium iodide) was added, and the cells were treated with trypsin and EDTA to be floated. The fluorescence at 565-640 nm emitted by the dead cells that had incorporated PI was measured by flow cytometry, and the cell number distribution was determined. In addition, without treating the cells with PI and trypsin, the concentration of lactic acid dehydrogenase in the culture supernatant was measured. As a result, adenosine strongly inhibited apoptosis, and the effect was clarified to have derived from a mitoKATP channel opening action via activation of the p38MAP kinase system (see non-patent reference 4).
Alternatively, (2) Griardi cells were seeded on a 6- or 24-well culture plate, cultured for a given time in a culture medium containing mitochondrial membrane potential sensitive dye JC-1, and then in a culture medium containing a mitochondrial depolarizing agent CCCP, and the change in the intensity of red fluorescence, which is an index of the mitochondrial membrane potential, was examined by flow cytometry. As a result, decreased fluorescence intensity and depolarization of mitochondria were observed (see non-patent reference 5).
Meanwhile, there is a report confirming a mitoKATP channel opening action of diazoxide in primary cultured rat neonatal cardiac muscle cell, utilizing the properties of ouabain, a cellular membrane Na/K ATPase inhibitor, to increase the intracellular Ca concentration and intramitochondrial Ca concentration (see non-patent reference 6).
However, the conventional methods are associated with aspects yet to be improved.
To be specific, in the aforementioned methods, (1) during cultivation under low oxygen conditions, the culture environment is difficult to maintain at a constant level and the levels of injury easily vary among wells; (2) cells need to be prepared in large amounts since cell injury is determined by flow cytometry or lactic acid dehydrogenase concentration measurement; (3) in flow cytometry, the operation up to a fluorescence detection is complicated; and (4) in the measurement of lactic acid dehydrogenase concentration, the measurement of absorbance is complicated.
On the other hand, a screening method of a compound influential on the oxidization-reduction potential of mitochondria, which is based on the measurement of the fluorescence change, has been reported (patent reference 1). Nevertheless, even by the method described in patent reference 1, evaluation of one sample requires a complicated operation and a considerable amount of operation time.
As described above, since conventional methods have difficulty in reproducing constant conditions, and since the time and labor necessary for the evaluation are enormous, the methods are not suitable for a simultaneous evaluation of a large number of pharmaceutical agents. patent reference 1: WO99/53024    non-patent reference 1: Kidney Int. 57, 838, 2000    non-patent reference 2: Circ Res. 84, 973, 1999    non-patent reference 3: Cardiovasc. Res. 55, 534, 2002    non-patent reference 4: Basic Res. Cardiol. 95, 243, 2000    non-patent reference 5: Cardiavasc. Res. 51, 691, 2001    non-patent reference 6: Circ. Res. 89, 856, 2001